The binding of the HIV envelope glycoprotein, gpl20, to its cell surface receptor, CD4, exposes novel "complex-dependent" gpl20 epitopes that elicit neutralizing antibody responses. These epitopes are situated in a gpl20 domain that binds 7 transmembrane spanning co-receptors needed for virus entry. Evidence that complex-dependent epitopes might be important vaccine targets was provided by our early studies showing: 1) that covalently crosslinked gp120-CD4 complexes elicit broadly neutralizing humoral responses against primary HIV isolates. In accordance, "fusion competent" immunogens containing gpl20 that is conformationally altered by cell surface receptor binding were recently shown to elicit broadly neutralizing humoral responses against a wide variety of HIV isolates from different genetic clades. These results warrant the development of vaccine strategies to raise humoral and cellular responses against complex dependent epitopes. Our hypothesis is that this can be accomplished by constructing single chain chimeric molecules comprised of gpl20 and CD4 covalently attached by a polypeptide linker. In this application, we provide preliminary evidence showing that such complexes are expressed from nucleotide sequences encoding codon-optimized gpl20 and soluble CD4. In order to evaluate our hypothesis, we propose to further develop and characterize these molecules, and evaluate their immunogenic properties in the contexts of DNA and subunit vaccines. The work will proceed with two specific aims. The first will include the production and characterization of constructs that express soluble, single-chain CD4-gpl20 complexes from M-tropic (R5), dual-tropic (R5X4) and T-tropic (X4) primary viruses. The second Aim will be to evaluate the immunogenicity of single chain CD4-gpl20 complexes in human CD4 transgenic mice. This work should provide new data with significant practical and scientific relevance to HIV vaccine development.